Inactivation of mTor: A Tool to Investigate Meiotic Progression and Translational Control During Bovine Oocyte Maturation

Summary

The protocol presented here describes the inhibition of the Ser/Thr kinase mTor during IVM of bovine oocytes. This approach can facilitate the investigation of meiotic progression and translational control. It also contributes to the definition of developmental competence and the improvement of IVM conditions.

Abstract

Although routinely used in breeding programs, in vitro maturation (IVM) of bovine oocytes and in vitro production (IVP) of embryos are nevertheless still the subject of basic research owing to suboptimal IVM conditions and variations in the developmental competence of the starting oocytes. In the present study we provide a method to inhibit the Ser/Thr kinase mTor during IVM using two independent inhibitors, Torin2 and Rapamycin. Both substances have different effects on meiotic progression and translational control and may allow discrimination between the mTorC1 and mTorC2 complex functions. The effects of the inhibitors are monitored by inspection of the chromatin configuration using aceto-orcein-staining as well as Western blotting and immunohistochemical analysis of the phosphorylation state of the translational repressor 4E-BP1, which is a prominent mTor target. Whereas Torin2 arrests bovine oocytes in the M I stage and inhibits 4E-BP1 phosphorylation, Rapamycin inhibits asymmetric division and does not influence 4E-BP1. Investigations utilizing these reactions can provide deeper insights into the regulatory events involved in meiotic maturation. Moreover, special focus can be placed on the temporal and spatial regulation of translational control. Such findings can contribute to the definition of the developmental competence of oocytes and to an improvement of IVM conditions.

Introduction

Fully grown bovine oocytes (arrested at prophase I; germinal vesicle -GV- stage) resume meiosis spontaneously when they are released from their follicles and transferred to a suitable culture medium. Previous investigations using the application of different inhibitors to in vitro culture media^{1, 2} revealed that activation of protein kinases and de novo protein synthesis trigger the maturation of mammalian oocytes and arrest them in metaphase II (M II), the stage suitable for fertilization. In the present study we describe a method to inhibit the Ser/Thr kinase mTorduring IVM of bovine oocytes. This approach might provide deeper insights into the complex processes involved in the regulation of protein synthesis in the context of meiotic maturation (transition from GV-stage to M II), because mTor links the phosphorylation of specific factors directly to translational control^{3, 4}.

The focus on the investigation of translational control reflects the importance of this process; fully grown oocytes are transcriptionally silent and protein synthesis relies on the activation of stored, dormant mRNAs⁵. In this context, mTor plays a predominant role. The kinase directly phosphorylates and inactivates repressors of the mRNA cap-binding protein eIF4E, the so-called 4E-binding proteins (4E-BP1-3), and thereby allows the formation of the 5´-mRNA-cap binding complex eIF4F (composed of eIF4E, the scaffold protein eIF4G and the RNA helicase eIF4A). Together with other factors it also stimulates ribosome binding and translation initiation⁶.

mTor, however, exists as two complexes: mTorC1 and mTorC2. Each complex is composed of different major regulators, differs in sensitivity to Rapamycin and has different cellular targets⁷. The major regulator of mTorC1, Raptor (regulatory-associated protein of mTOR), phosphorylates components of the translational machinery, namely ribosomal proteins (for instance RPS6 at Ser235/36) and the translational repressor 4E-BP1 (at Thr37/46/65/70). The major regulator of mTorC2, Rictor (Rapamycin-insensitive companion of mTOR), is Rapamycin- resistant and phosphorylates Akt (PKB) which in turn phosphorylates mTorC1. Preliminary investigations in bovine oocytes during IVM revealed different transient activities of mTorC1 and mTorC2 during IVM. In the GV-stage of oocytes the mTorC2 is active³ ; it is inactivated in the course of IVM. In contrast, mTorC1 shows the opposite behavior³. These results correspond with findings showing that 4E-BP1 phosphorylation is lower in the GV-stage, continuously increases during IVM, and is highest in the M II stage^{8, 9}.

However, mTorC1 and C2 both respond to the active site inhibitor Torin2¹⁰ and might have other (yet unknown) targets. Candidates are meiotic spindle-forming or regulatory proteins, since mTor associates with meiotic spindles during chromatin segregation.

From a practical point of view, it should be noted that in vitro systems yield only 30-40 % transferable embryos in the bovine species ¹¹. The causes for this could be suboptimal in vitro conditions and/or differences in the developmental competence of the starting oocytes which occur despite their selection from follicles of a defined size. However, detailed investigations of meiotic maturation on a molecular level can contribute to the optimization of IVM systems. Furthermore, oocytes might be selected according to their developmental competence, for instance by IVM systems under inhibitory conditions (see discussion). Hence, in the procedure presented here, we used two independent mTor inhibitors, Torin2 and Rapamycin, which resulted in different chromatin statuses and differential phosphorylation of 4E-BP1. Interestingly, approximately 20 % of the oocytes overcame the Torin2 block and might thus be candidates which possess a high developmental competence.

Protocol

All experiments were performed in accordance with the guidelines of the local ethics committee (Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei, Mecklenburg-Vorpommern, Germany). The ovaries were obtained from a commercial slaughterhouse. The remaining carcass was used for meat production.

1. Solutions to be prepared.

1. Prepare PBS for gaining oocytes by weighing Glucose (500 mg), Pyruvate (18 mg), Penicillin (10 mg), Streptomycin (20 mg), Heparin (5.6 mg) and BSA (150 mg) and add these substances to 500 mL PBS pH 7.4, supplemented with 1mM Ca²⁺/Mg²⁺. Store the solution until needed at 4°C (up to 2-3 days).
2. Prepare TCM as the maturation medium by weighing NaHCO₃ (220 mg), Gentamycin (5 mg), Pyruvate (2.2 mg) and TCM-199 Hepes modification (1510 mg) and add those substances to 100 mL ddH2O. Mix the medium for 40 min on a magnetic stirrer. Adjust the pH-value to 7.4. Use a sterile filter to fill the solution into a small, sterile bottle. Store the solution until needed at 4°C (up to 2 weeks).
3. Prepare the Rapamycin working solution with a concentration 800 µM by dissolving 18.2 µg Rapamycin in 25 µL DMSO. Store the solution in aliquots of 5 µL at -20°C.
4. Prepare the 20 mM Torin2 stock solution by dissolving 10 mg Torin2 in 1.16 mL DMSO. Store aliquots of 10 µL at -20°C. Just before performing an experiment, take 3 µL of this stock solution and dilute it in 247 µL DMSO to prepare a working solution with a concentration of 240 µM.
5. Prepare 10 mL of the PBS as a washing buffer for immunohistochemistry. Supplement PBS with 0.5 % BSA and 0.05 % Triton-X-100.
6. Prepare 10 mL of the permeabilization buffer by weighing 0.46 g Hepes, 10.27 g sucrose, 0.29 g NaCl, 0.061 g MgCl₂ and 0.5 mL Triton-X-100. Fill up with ddH₂O and adjust to pH 7.4.
7. Prepare 10 mL of the blocking solution (BS) by diluting the commercial blocking solution 1:50 with ddH₂O.
8. Prepare the working solution for antibody incubation by diluting the BSsolution with PBS, pH 7.4, 0.05 % Tween (1:1).
9. Fix and store the oocytes in 3 % paraformaldehyde, 2 % sucrose.

2. Source of oocytes and preparations before going to the slaughterhouse.

1. Prepare a NaCl-solution (0.9 %) by dissolving 9 g NaCl in 1 L ddH₂O. Warm this solution up to 37°C and fill it into a thermally insulated vessel.
2. Recover the ovaries at the slaughterhouse.Remove the ovaries with a pair of scissors from the carcasses of healthy cows immediately after the abdominal cavity is opened by a knife cut of about 40 cm in the ventral median line. Put the ovaries into the vessel with the NaCl-solution. Transport the ovaries back to the laboratory within 3 h after slaughter.
3. Puncturing of the follicles and gaining of the cumulus-oocyte complex's (COCs).
   1. Use a 5 mL syringe to aspirate 1 mL warm PBS (37°C) first and then puncture the follicles (estimated follicle diameter: 4-8 mm). Remove the needle from the syringe and transfer the liquid in a plastic tube (50 mL) with a screwtop and keep it warm. Repeat the previous steps until all follicles sized 4-8 mm are aspirated.
   2. Allow the aspirated liquid to stand for 10 min. The developing sediment contains approximately 400 COCs aspirated from 50 to 80 ovaries.
   3. Use a Pasteur pipette to transfer sedimented COCs into screened Petri dishes with a diameter of 90 mm. Add 5 mL PBS to each of the Petri dishes and store the dishes on a hotplate (37°C).
   4. Take the COCs out of the Petri dishes with a pipette controller (use micropipettes with a volume of 20 µL) with the help of a stereomicroscope with 15x magnification and transfer them to smaller Petri dishes (diameter of 30 mm) filled with PBS.
4. Classification of the COCs¹²
   1. Use only COCs categorized as oocytes with a dark, homogenous cytoplasm and a compact (or only slightly expanded) cumulus oophorus that shows more than five layers of cumulus cells.

3. In vitro maturation and inhibitor treatment.

1. Preparing the maturation dishes.
   1. Weigh 60 mg BSA and add it to 20 mL TCM and put it into the incubator for at least 1 h at 39°C, 5 % CO₂ and saturated humidity. Use a syringe with a sterile filter to distribute the TCM equally into two 30 mm Petri dishes.
   2. Transfer 400 µL TCM from the two Petri dishes in each well of four 4-well dishes (these are the washing dishes), so that an equal amount of TCM remains in the Petri dishes.
   3. Take 2760 µL of TCM from one Petri dish and add 40 µL of Rapamycin (800 µM) or Torin2 (240 µM)-working solution (these are the treatment media). Take 2760 µL of TCM from the other Petri dish and add 40 µL of DMSO (this is the control medium).
   4. Add 350 µL of the treatment media and the control medium to each well of two 4-well dishes.
   5. Transfer 25 COCs from the Petri dishes with PBS from point 2.3.5 / 2.4.1 after classification and put them into each well of the four 4-well washing-dishes.
   6. Transfer the COCs from two 4-well-washing-dishes with a pipette using a volume of 50 µL to the two 4-well-dishes containing Rapamycin/Torin2 working solution, so that the total volume in each well is 400 µL and the final concentration is 3 µM for Torin2 and 10 µM for Rapamycin.
   7. Do the same with the other washing dishes and the 4-well dishes containing DMSO for the control group. Put the dishes with the COCs into the incubator at 39°C, 5 % CO₂ and saturated humidity. Let the COCs mature there for 24 h.

4. Morphological inspection by Aceto-Orcein staining.

1. Fixation of the oocytes.
   1. Prepare two 4-well-dishes with 400 µL PBS per well.
   2. Transfer 100 oocytes of the Torin/Rapamycin group to well 1 of one of the 4-well dishes with PBS and 100 oocytes of the control group to the other well.
   3. Denude the oocytes by repeated pipetting with 200 µL pipette tips so that the cumulus cells are completely removed. Use the remaining 3 wells to wash the oocytes.
   4. Prepare microscope slides as follows:
      1. Apply two vertical parallel lines of silicone of medium viscosity with the help of a needle attached to a 2 mL-syringe spaced about 1.5 cm apart to each side of the microscope slide.
      2. Use a pipette to transfer 10 oocytes in about 4 µL PBS to the microscope slide. NOTE: The drop with the oocytes should be placed in the center between the two lines of silicone at one side of the microscope slide. Take a microscope cover glass (18x18 mm) and place it carefully onto the lines of silicone and the drop with the oocytes in between.
      3. Press the cover glass lightly and carefully so that the oocytes do not burst. Put a drop of ethanol-glacial acetic acid (3:1) at one edge of the cover glass, so that the liquid may flow through the whole space in between cover glass and microscope slide and enclose the oocytes completely.
      4. Put the microscope slides in a cuvette filled with 40 mL ethanol (absolute) glacial acetic acid (3:1). Ensure that the lower edges of the cover glasses are immersed in the liquid.
      5. Seal the cuvette air tight with self-sealing lab film and store it at 4°C until staining. Keep the slides for no more than two weeks.
2. Staining of the oocytes.
   1. Take the microscope slides out of the cuvette and dab them gently with a tissue. Apply 4 drops (approx. 40 µL) of Aceto-Orcein-solution (2 %) to the upper edge of one cover glass so that the liquid is soaked under the cover glass.
   2. Place a strip of filter paper at the lower edge of the cover glass to get the liquid completely soaked through the space between microscope slide and cover glass. Let the staining solution incubate for 10 min.
   3. Apply drops of acetic acid (30 %) to the lower edge of one cover glass and use a strip of filter paper at the upper edge of the cover glass to get the liquid soaked through until the interstice is completely clear.

5. Evaluation.

1. Examine the stained slides under a phase-contrast microscope at 100-400x magnification. Classify the oocytes by reference to the chromatin configuration in metaphase II, anaphase II, telophase II, metaphase I, GVBD-stage, GV-stage and degenerate oocytes^1,5.

6. Western blotting was performed according to standard procedures¹³.

1. Subject 50 oocytes per lane to SDS-PAGE on 13 % (w/v) acrylamide gels with an acrylamide: bisacrylamide ratio of 30:0.8. After transferring, block membranes with 5 % fat-free dry milk powder in TTBS for 1 hour at room temperature.
2. Dilute the primary antibodies as follows: 4E-BP1, 1:800; p4E-BP1-Thr37/46, 1:500; RPS6, 1:1000; pRPS6-Ser235/36, 1:1000. NOTE: In all cases, the HRP-labeled secondary antibody is four times more highliy diluted than the primary. Perform ECL detection with a commercial kit.

7. Immunohistochemistry and Confocal Laser Scanning Microscopy (LSM)

1. Wash the fixed oocytes (1.9) once with PBS washing solution at room temperature. Incubate for 5 min in permabilization solution at 0°C. Wash four times, 15 minin washing buffer. Incubate 2 h in blocking solution at room temperature.
2. Incubate the primary antibody (4EBP1, 1:100; p4E-BP1 Ser37/46, 1:200) overnight at 4°C without washing. Wash four times, 15 minin washing buffer. Incubate the secondary, fluorescence dye labeled antibody (1:200) for 5 h in the dark at room temperature. Wash four times, 15 minin washing buffer.
3. Stain the chromatin with SYBR green in the dark for 1 h. Wash four times, 15 minin washing buffer. Fix the oocytes in 2 % paraformaldehyde overnight at 4°C. Mount the oocytes in glycerin gelatin on microscopy slides.
4. Perform fluorescence analysis using a confocal laser scanning microscope. Track1: Laser: Argon 30mW (458/488/514nm) 488nm/12%; HFT/NFT: 488/545nm; Filter: BP 505-530nm Channel1 (Channel2 closed). Track2: Laser: HeNe 1mW (543) 543/60%; HFT/NFT: 543/545nm; Filter: LP 560-nm Channel2 (Channel1 closed). Pinhole: 1AU, gain constant, veraging: 2, Resolution 1024x1024. Stacks: 1 µm between slices.

תוצאות

Source of oocytes, in vitro maturation and mTor inhibition

Figure 1 illustrates the collection of cumulus oocyte complexes (COCs) and the analysis of the chromatin configuration. Only healthy ovaries (Figure 1A) obtained from a local slaughterhouse were used as oocyte sources. COCswere aspirated from follicles sized 4-8 mm (Figure 1B). Only COCs with compact layers of cumulus cells (Figure 1C) were used fo...

Discussion

In vitro maturation (IVM) of bovine oocytes is an important technique because it is an integral part of the in vitro production of embryos in specific breeding programs. However, a critical limitation is the fact that no method exists to assess the developmental competence of fully grown oocytes directly after follicular release. Furthermore, when the chromatin status of bovine oocytes is analyzed morphologically, more than 90 % have reached the M II stage after 24 h of IVM under standard conditions1. However, transfera...

Materials

Name	Company	Catalog Number	Comments
PBS Dulbecco wCa2+ wMg2+	Biochrom AG	L1815	For gaining of the COC and denudation
D-(+)-Glucose	Sigma	G5400-250G	Supplement to PBS Dulbecco
Sodium pyruvate	Sigma	P3662-25G	Supplement to PBS Dulbecco
Penicillin G sodium salt	Sigma	P3032-25 MU	Supplement to PBS Dulbecco
Streptomycin sulfate salt	Sigma	S1277-50G	Supplement to PBS Dulbecco
Heparin sodium salt	Sigma	H3149-25 KU	Supplement to PBS Dulbecco
Bovine serum albumin	Sigma	A9647-10G	Supplement to PBS Dulbecco
Sodium bicarbonate (NaHCO3)	Sigma	S4019-500G	Supplement to PBS Dulbecco
Gentamicin sulfate salt	Sigma	G3632-250MG	Component of TCM medium
TCM-199 Hepes Modification	Sigma	M2520-1L	Component of TCM medium
Sodium chloride (NaCl)	Sigma	S5886-500G	Component of 0.9% NaCl solution
Rapamycin	Cell Signaling Technology (CST)	9904 S	m-Tor-inhibitor
Dimethyl sulfoxide (DMSO)	Serva	39757.01	Used as solubilizer for Rapamycin and Torin 2
Torin 2	R&D Systems	4248/10	m-Tor-inhibitor
Phosphat Buffered Saline Tablets (PBS)	Sigma	P-4417	for PBS-präparation
Tween 20	Serva	39796.01	permeabilization buffer
Roti-Immuno-Block	Roth	T144.1	Blockingsolution+Lsg.für AK
Triton X100	Serva	37240	permeabilization buffer
Hepes	Serva	25245	permeabilization buffer
Albumin Fraktion V(biotinfrei)	Roth	0163.3	permeabilization buffer
D(+)-Saccharose	Roth	4621.1	permeabilization buffer
Nacl	Roth	3957.1	permeabilization buffer
MgCl2x6 H2O	Sigma	M2393	permeabilization buffer
Paraformaldehyde	Sigma	P-6148	Fixation
Kaisers Glyceringelatine	Merck	109,242	cover oocytes
Alex-Fluor  546F(ab)2 fragment goat anti rabbit IgG( H+L)	MOBITEC	A11071	secondary antibody fluorecent labeled
SYBR Green nucleic acid gel stain	Invitrogen	S7563	DNA staining
4E-BP1	CST	9452	primary antibody
p4E-BP1 Thr37/46	CST	2855	primary antibody
anti rabbit IgG-HRP	CST	7074	secondary antiboday HRP labelled
ECL prime	GE healthcare	RPN2232	WB detection
Fluid filter Infufil 0,2µm 5,7 cm2	Fresenius Kabi	2909702	Used for sterile filtration
Pipette pipetman P10 0,1-10µl	Gilson		Used for transferring denudated oocytes and embryos and volumina of 0,1-10µl
Pipette Reference 10-100µl	Eppendorf	4920000059	Used for transferring COC and volumina of 10-100µl and for denudation
Pipette Reference 100-1000µl	Eppendorf	4920000083	Used for transferring volumina of 100-1000µl
Micro-classic pipette controller	Brand	25900	Used for transferring COC
Micropipettes intraMark 20µl	Brand	708718	Used for transferring COC
Safe-lock tubes 0,5 ml	Eppendorf	0030 121.570	Used to store volumina up to 0,5 ml
Polypropylene centrifuge tube with conical base 50 ml, 30,0/115mm	Greiner	210261	Used for the aspirated fluid to sediment
Pasteur pipette 7 ml	VWR	612-1681	Used for transferring the sediment to screened Petri dishes
Germ count dish with vents 90/16 mm	Greiner	633175	Used for searching the COC
Petri dish with vents 35/10 mm	Greiner	627102	Used for medium preparation, washing the COC
Petri dish with vents 60/15 mm	Greiner	628102	Used for medium preparation
Multidish 4 wells	Thermo Scientific	176740	Used for washing the COC, for COC maturation, embryo culture and for denudation of the oocytes
Tissue culture dish 35/10 mm, 4 compartments	Greiner	627170	Used for washing the COC
Microscope slides 76x26x1mm	Thermo Scientific	AB00000112E	For fast morphological inspection by Aceto-Orcein-staining
Microscope cover glasses, 18x18 mm	VWR	ECN 631-1567	For fast morphological inspection by Aceto-Orcein-staining
Needle 18G x 1 1/2"  1,2 x 40 mm	BD Microlance	REF 304622	For medium preparation
Incubator inc108med with CO2 control	Memmert	84198998	Used for in vitro maturation
Universal oven model UNB 200	Memmert	84193990	Used for medium preparation
Control unit HAT 400 W1, 72VA 470 x 263 mm	Minitube	12055/0400	Used for keeping the cells warm in the course of treatment
confocal laser scanning microscope, phase contrast microscope	Zeiss	Model: LSM 5 PASCAL Axiovert 200 M	Morphological and immuno-histochemical analysis of oocytes